The DNA damage response (DDR) is a coordinate set of events that promptly follows the generation of a lesion in the DNA double helix. Detection of DNA discontinuities by specialized factors initiates a signaling cascade that, stemming from the site of DNA damage, amplifies the signal and reaches the whole nuclear space and the entire cell1. DDR signaling cascade initiation establishes a local self-feeding loop that leads to focal accumulation of upstream DDR factors in the form of cytologically detectable DDR foci at damaged sites. Specifically, detection of a DNA double-strand break (DSB) triggers the activity of the protein kinase ATM that, among other factors, phosphorylates the histone variant H2AX (γH2AX) at the DNA damage site. This modification recruits DDR-mediators like MDC1 and 53BP1 that boost ATM activity. DDR activation can be triggered by exogenous DNA damaging agents such as ionizing radiations and chemotherapeutic agents (i.e. including but not limited to bleomycin) and by endogenous physiological events such as meiotic recombination, V(D)J recombination at the immunoglobulins and T cell receptor loci, telomere shortening and reactive oxygen species, as well as pathological events such as oncogene activation, viral integration in the genome, viral replication and bacterial infection1,82. Telomeres dysfunction and oncogene activation can generate a sustained DDR leading to a permanent cell-cycle arrest known as cellular senescence2. Recently also bacteria have been shown to generate persistent DNA damage and cellular senescence in mammals82. Several pathologies associated with altered telomere functions have been reported as “telomeropathies”85.
It has recently been appreciated that mammalian genomes are pervasively transcribed and the vast majority of DNA sequences can be found in primary, often overlapping, transcripts most of which apparently not associated with coding functions3. These non-coding RNAs (ncRNAs) may remain associated with chromatin, and some aggregate in subnuclear structures such as speckles and paraspeckles4. An unsuspected increasing number of these ncRNA transcripts have been shown to be evolutionarily conserved among related species5,6 and play a variety of relevant cellular functions by regulating the localization and the activity of proteins and/or providing structural support for cellular and sub-cellular structures7 and controlling chromatin-modification4,8 and enhancer-like functions9. These activities may be exerted despite estimated very low levels of expression, few molecules per cell, for some of these RNA molecules10,11,12,13. Some ncRNAs may be processed by ribonucleases of the RNA interference (RNAi) pathway, giving rise to short double-stranded RNA products that participate in various cellular functions. The RNAi pathway is a conserved machinery, whose components are thought to have evolved to preserve genome integrity from the attacks of viruses and mobile genetic elements14. It involves different types of short double-stranded RNA molecules including small interfering RNAs (siRNAs), microRNAs, repeat-associated small interfering RNAs (rasiRNAs), Piwi-interacting RNAs (piRNAs)15 and QDE-2 interacting RNAs (qiRNA) in Neurospora crassa16. It is commonly thought that only microRNA maturation is dependent on both DROSHA and DICER endonucleases, two RNase type III enzymes that process hairpin structures to generate double-stranded microRNAs17. In mammals, microRNAs modulate gene expression usually by their ability to regulate mRNA translation and stability and have been involved in several processes such as cell fate determination, transformation, proliferation and cell death18. piRNAs and qiRNAs have been implicated in genome stability maintenance16 and a family of microRNAs (miR-34) has been shown to act downstream of p5319. It is presently unknown whether any RNAs have any direct role in the control of DDR activation at sites of DNA damage.
US2006105384 is focused on a technique for detecting and diagnosing disease conditions, as well as health conditions due to exposure to environmental conditions by detecting and identifying DNA or RNA damage markers. This technique is based on measurement of free levels of nucleotide excision products resulting from DNA or RNA damage. The DDRNAs of the instant invention are not nucleotide excision products.
JP2009171895 concerns a method for analyzing the function of a non-coding RNA (ncRNA) existing in a nucleus by destroying the ncRNA by introducing an antisense oligo-molecule containing substantially the same sequence as a sequence complementary to a single-stranded region in the secondary structure of the target ncRNA to a cell nucleus and destroying the RNA molecule.
WO2012/013821 relates to the field of cancer, particularly cancers wherein p53 tumour suppression function is lost or impaired. It is shown herein that Dicer is a synthetic lethal partner of p53, allowing the selective targeting and killing of cancer cells. The effects of Dicer on survival on cancer cells are mediated through the miR17-92 cluster and inhibition of members of this miRNA cluster is an attractive treatment strategy in cancer. Most particularly, these findings are of importance in the field of retinoblastoma.
WO2011/157294 relates to compositions comprising an inhibitor of a polynucleotide, said polynucleotide to be inhibited being capable of decreasing or suppressing expression of Dicer or a biologically active derivative thereof for use in treating or preventing cancer, metastasis, heart failure, cardiac remodelling, dilated cardiomyopathy, autoimmune diseases, or diseases or disorders related thereto. Furthermore, the present invention also relates to methods of treating or preventing cancer, metastasis, heart failure, cardiac remodelling, dilated cardiomyopathy, autoimmune diseases, or diseases or disorders related thereto. DDRNAs are not mentioned nor the impact of Dicer modulation on DNA damage related events and DDR modulation.
WO2009/102225 relates to compositions and methods for cancer diagnosis, research and therapy, including but not limited to, cancer markers. In particular, the present invention relates to ncRNAs as diagnostic markers and clinical targets for prostate, lung, breast and pancreatic cancer.
US2012289581 relates to long non-coding RNAs (lncRNAs) and methods of using them diagnostically and therapeutically for treatment of cancer, stem cell therapy, or regenerative medicine are disclosed. In particular, the invention relates to lncRNAs that play roles in regulation of genes involved in cell proliferation, differentiation, and apoptosis. Such lncRNAs can be used as biomarkers to monitor cell proliferation and differentiation during cancer progression or tissue regeneration. One of the identified lncRNAs, referred to as PANDA (a P21-Associated NcRNA, DNA damage Activated), inhibits the expression of apoptotic genes normally activated by the transcription factor NF-YA. Inhibitors of PANDA sensitize cancerous cells to chemotherapy and can be used in combination with chemotherapeutic agents for treatment of cancer.
Limmer K et al. (2013) used a Molecular Force Assay (MFA) to measure the activity of Dicer. As a model system, they used an RNA sequence that forms an aptamer-binding site for paromomycin, a 615-dalton aminoglycoside. They have shown that Dicer activity is modulated as a function of concentration and incubation time: the addition of paromomycin leads to a decrease of Dicer activity according to the amount of ligand. The measured dissociation constant of paromomycin to its aptamer was found to agree well with literature values. The parallel format of the MFA allows a large-scale search and analysis for ligands for any RNA sequence.
Wei et al, (2012) reports the existence in plants and in a human cancer cell line of small RNAs, named diRNAs, generated in proximity to DNA DSB sites81. The authors show that genetic inactivation of Dicer-like RNA endonucleases results in a specific defect in DNA repair by homologous recombination. Authors observe some correlation between diRNAs accumulation and DNA repair by homologous recombination, and propose that diRNAs control DNA repair. However there is no support by the data shown in the article by Wei et al. to such hypothesis. As a matter of fact there is no evidence that diRNA play a biologically active role in the process of DNA repair. Prior art data in Wei et al are not in contrast with diRNAs being generated following the degradation of a RNA transcript spanning the DSB site.
In addition, it is not demonstrated in the Wei et al article that the proposed effect of the inactivation of Dicer-like genes and DNA repair is not indirect, possibly mediated by canonical RNA interference mechanisms. Although the authors show that the abundance of few DNA repair factors is not affected, it is not demonstrated that other DNA repair factors, not tested by the authors, are unaffected and not targeted by RNA interference mechanisms and thus potentially making an indirect impact on DNA repair.
Finally, correlation is not always maintained and at least in plants the authors show cases in which diRNAs are decreased (FIG. 3a, mutants RDR2 and RDR6) and DNA repair is unaltered (FIG. 3b).
DDRNA of the instant invention have been characterized for distinct functions: DDRNAs control DDR signaling, whereas diRNA of Wei et al are not shown to have any role in DDR signaling: Wei et al show no evidence of altered DDR activation, as detected by nuclear DDR foci formation or of DDR proteins activation, for instance by phosphorylation, or of altered DNA damage checkpoint functions or modulation of cellular senescence. Thus there is no demonstrated overlap between their functions.
In cultured Drososphila cells, Michalik et al.79, showed that the transfection of a linearized plasmid leads to the generation of short (21 nt) RNAs with the sequence of the plasmid DNA ends. The small RNAs in this system are produced by active transcription of plasmid genes in the vicinity of the break. The function proposed for them was the repression of the marker gene encoded by the plasmid. Inactivation of some of the factors involved in the RNA interference pathway relieves the observed repression. Such effect has been interpreted as RNA interference activity of the short RNAs acting as endo-siRNAs. A causal relation between the production of short RNA and DDR activation or DNA repair is lacking in this study. This set of observation support the notion that small RNA are produced at DNA ends in cultured Drosophila cells, but it does not provide a function of this novel RNA molecule in the DNA damage response pathway.